Up-regulated FHL1 expression maybe involved in the prognosis of Hirschsprung's disease.

BACKGROUND: In a subset of patients with Hirschsprung's disease (HSCR), gastrointestinal motor dysfunction persisted long after surgical correction. Gastrointestinal motility is achieved through the coordinated activity of the enteric nervous system, interstitial cells of Cajal, and smooth muscle (SMC) cells. Inhibition of four-and-a-half LIM protein-1 (Fhl1) expression by siRNA significantly decreases pulmonary artery SMCs migration and proliferation. Furthermore when up-expressing FHL1 in atrial myocytes, K (+) current density markedly increases, therefore changing myocytes' response to an electrical stimulus. However whether FHL1 in colon SMCs (the final effector organ) influences intestinal motility in HSCR patients has not been clarified. METHODS: FHL1 mRNA and protein expressions were analyzed in 32 HSCR colons and 4 normal colons. RESULTS: Smooth muscle layers were thicken and disorganized in HSCR. FHL1 was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the ganglionic colon. FHL1 mRNA relative expression level in aganglionic colons was 1.06 ± 0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83 ± 0.09. FHL1 expression level in ganglionic colon (1.66 ± 0.30) or aganglionic colon (1.81 ± 0.35) was significantly higher than that in normal colons (P=0.045 and P=0.041, respectively). Meanwhile, we found FHL1 expression in aganglionic colon was slightly stronger than that in ganglionic colon (P=0.036). CONCLUSION: These data suggested that up-regulated FHL1 in smooth muscle in HSCR might be associated with intestinal wall remodeling in HSCR and might be one of the risk factors for gastrointestinal motor dysfunction.

The role of CD44-hyaluronic acid interaction in exogenous mesenchymal stem cells homing to rat remnant kidney.

BACKGROUND/AIMS: The aim of our study was to reveal the role of CD44-Hyaluronic acid (HA) in the homing and improving renal function of systemically transplanted MSCs in chronic renal failure. METHODS: First, a remnant kidney model was established in rats and the expression of HA was determined using immunohistochemistry (IHC) and western blotting. Next, chemotaxis assay using flow cytometry, and cell migration assay of MSCs were performed in vitro. Then, MSCs were transplanted into rats, thus, sprague-Dawley (SD) rats were randomly divided into sham group, 5/6 nephrectomy (5/6 Nx) group, MSC group and MSC/Anti-CD44 group (n = 8 for all groups). Migration of MSCs to the kidney in these rats was assessed by using cell tracking experiments, and tissue damage was evaluated by morphological analysis using Masson's trichrome staining and periodic acid Schiff staining. RESULTS: HA was significantly observed in 5/6 Nx group, but not in sham group. Meanwhile, HA was discovered induced MSCs migration remarkably (p < 0.05) and anti-CD44 antibody inhibited the migration significantly (p < 0.05) in vitro. In vivo, the GFP-MSCs were observed in MSC group and the cells reduced in MSC/Anti-CD44 groups, especially, in the tubulointerstitium. CONCLUSION: Our findings reveal that CD44-HA has the potential to induce MSCs homing to injured tissue, while its effect on the ability of MSCs, improving tissue function, is not significant.

SEMA3A rs7804122 polymorphism is associated with Hirschsprung disease in the Northeastern region of China.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of intrinsic ganglion cells in the nerve plexuses of the lower colon. Our previous results showed increased semaphorin 3A (SEMA3A) expression may be the risk factor for HSCR pathology in a subset of patients. Therefore, the association between polymorphisms in SEMA3A and the risk of HSCR was examined. METHODS: The genotypes of two SNPs (rs7804122 and rs797821) in the SEMA3A gene in 119 patients with HSCR and 93 controls were examined using PCR-sequencing to determine the contribution of SEMA3A to the HSCR phenotype. PCR reaction with cDNA template was also used to find out whether a novel mutation (Chr7:83634610A→T) influences the SEMA3A pre-mRNA splicing. RESULTS: Genotypes comprising allele G of rs7804122 (GG or AG) were over-represented in patients (48.74 vs. 24.8%; p = 0.0013) which indicated that the risk of HSCR was significantly higher among subjects with the GG or AG genotype than among the subjects with the AA genotype. No statistically significant associations were found for SNP rs797821 at the allele or genotype levels. The differences in genotypes and allele distributions of rs7804122 and rs797821 between various clinical classifications were not statistically significant. The novel heterozygous mutation (Chr7:83634610A→T) 30bp away from an intron/exon boundary, had no detectable effect on splicing efficiency. CONCLUSION: Our results for rs7804122 provided preliminary evidence that the SEMA3A gene is involved in the susceptibility to HSCR in the Northeastern Chinese population.

Semaphorin 3A expression in the colon of Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of intrinsic ganglion cells in the nerve plexuses of the lower colon. The Semaphorin 3A (SEMA3A) gene is involved in the migration of enteric neural precursors (ENPs). To analyze the function of SEMA3A in HSCR, the SEMA3A expression in different colon segments in HSCR was examined. METHODS: The expression levels of SEMA3A in both ganglionic and aganglionic colon tissues of 32 patients with HSCR and in colon tissue of 5 newborn unaffected individuals were examined by real-time RT-PCR, Western-blot, and immunohistology. RESULTS: Comparison of SEMA3A expression levels between ganglionic and aganglionic tissues in HSCR revealed upregulation of SEMA3A expression in 43.75% (14/32) of the aganglionic colons. SEMA3A was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the normal colon of both unaffected newborns and patients with HSCR. In the aganglionic segment of patients with HSCR, SEMA3A was highly expressed in the circular muscle layer and was also detected in the submucosa and in the longitudinal muscles layer. The fluorescence intensity of SEMA3A in the circular muscle layer in the aganglionic segment was much higher than that in ganglionic segment (p < .001). CONCLUSION: SEMA3A expression was upregulated in the aganglionic smooth muscle layer of the colon in some patients with HSCR and our data suggest that increased SEMA3A expression may be a risk factor for HSCR pathology in a subset of patients.

Cordyceps sinensis attenuates HBx­induced cell apoptosis in HK­2 cells through suppressing the PI3K/Akt pathway.

The authors' previous studies demonstrated that the major renal damage from hepatitis B virus infection is HBx­induced apoptosis of renal tubular epithelial cells. Cordyceps sinensis is one of the most valuable of traditional Chinese medicines and is extensively used to treat chronic renal diseases. However, there is no research on the potential renal protective effect of C. sinensis on HBx­induced apoptosis of renal tubular cells. The protective effect and underlying mechanism of C. sinensis were examined using a renal tubular epithelial cell line stably overexpressing HBx. HK­2 cells were stably transfected with pCMV­HBx to establish HBx­overexpression in an in vitro cell model and HK­2 cells transfected with an empty vector were generated as a control. The effect of C. sinensis on cell proliferation and apoptosis, the phosphatidylinositol­3­kinase (PI3K)/protein kinase B (Akt) signaling pathway, and the enzyme activity of caspase­3 and caspase­9 was measured. The present study demonstrated that HBx transfection inhibited cell proliferation; increased apoptosis, caspase­3 and caspase­9 activity; and increased the activity of the PI3K/Akt pathway. Treatment with C. sinensis attenuated all of these HBx­induced responses. HBx triggered apoptosis and activated the PI3K/Akt signaling pathway in HK­2 cells. C. sinensis treatment significantly attenuated the effect of HBx, at least in part by suppressing the PI3K/Akt signaling pathway.

HDAC-selective Inhibitor Cay10603 Has Single Anti-tumour Effect in Burkitt's Lymphoma Cells by Impeding the Cell Cycle.

Histone deacetylases (HDACs) inhibitors are novel in cancer therapy nowadays. HDAC6-selective inhibitors exert advantageous effects due to higher selectivity and less toxicity. We explored the anti-tumor effect and the molecular mechanism of cay10603, a potent HDAC6 inhibitor in Burkitt's lymphoma cells. Our study revealed cay10603 inhibited the proliferation of Burkitt's lymphoma cell lines, and induced caspase-dependent apoptosis. Cay10603 inhibited the expression of CDKs and cyclins to impede cell cycle progression in both Burkitt's lymphoma cell lines. Cay10603 also showed the additive effect with vp16 notably. Our data presented the promising anti-tumor effect of cay10603 in the Burkitt's lymphoma therapy.

Cordyceps sinensis attenuates renal fibrosis and suppresses BAG3 induction in obstructed rat kidney.

BAG3 regulates a number of cellular processes, including cell proliferation, apoptosis, adhesion and migration, and epithelial-mesenchymal transition (EMT). However, the role of BAG3 in renal tubular EMT and renal interstitial fibrosis remains elusive. This study aimed to examine the dynamic expression of BAG3 during renal fibrosis, and to investigate the efficacy of Cordyceps sinensis (C. sinensis) on renal fibrosis. A rat model of unilateral ureteral obstruction (UUO) was established, and the expression of BAG3 and α-SMA, and the efficacy of C. sinensis on renal fibrosis induced by UUO were examined. The results showed that UUO led to collagen accumulation, which was significantly suppressed by C. sinensis. UUO increased the expression of BAG3 and α-SMA, a mesenchymal marker, while UUO induced BAG3 and α-SMA expression was significantly inhibited by C. sinensis. In addition, immunohistochemical staining demonstrated that BAG3 immunoreactivity was restricted to tubular epithelium. In conclusion, BAG3 is a potential target for the prevention and/or treatment of renal fibrosis, and C. Sinensis is a promising agent for renal fibrosis.

Dynamic expression of molecules that control limb muscle development including Fhl1 in hind limbs of different gestational age.

Muscle abnormality could be a key reason for congenital clubfoot (CCF) deformity, which manifests itself during fetal development. FHL1 down-regulated expression is involved in the formation of skeletal muscle abnormalities in CCF and FHL1 gene mutations contribute to the development of some kinds of myopathies. Therefore, detecting dynamic expression of Fhl1 and other molecules (Hgf, MyoD1, Myogenin, and Myh4) that control limb muscle development in hind limbs of different gestational age will provide a foundation for further research on the molecular mechanism involves in the myopathies or CCF. The dynamic gene expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 in the lower limbs of E16, E17, E19, and E20 rat embryos were examined by real-time RT-PCR. Immunofluorescence was used to detect formation of specific muscle fibers (fast or slow fibers) in distal E17 hind limbs. The expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 were varying in hind limbs of different gestational age. Real-time PCR results showed that all the genes that control skeletal muscle development except for Fhl1 exhibited a peak in E17 lower limbs. Immunofluorescence results showed obviously positive fast-myosin in the distal E17 lower limbs and meanwhile slow-myosin had no apparently signals. E17 was a critical time point for terminal skeletal muscle differentiation in the lower limbs of rat embryos.

Altered expression of 14-3-3ζ protein in spinal cords of rat fetuses with spina bifida aperta.

BACKGROUND: A large number of studies have confirmed that excessive apoptosis is one of the reasons for deficient neuronal function in neural tube defects (NTDs). A previous study from our laboratory used 2-D gel electrophoresis to demonstrate that 14-3-3ζ expression was low in the spinal cords of rat fetuses with spina bifida aperta at embryonic day (E) 17. As a member of the 14-3-3 protein family, 14-3-3ζ plays a crucial role in the determination of cell fate and anti-apoptotic activity. However, neither the expression of 14-3-3ζ in defective spinal cords, nor the correlation between 14-3-3ζ and excessive apoptosis in NTDs has been fully confirmed. METHODOLOGY/PRINCIPAL FINDINGS: We used immunoblotting and quantitative real-time PCR (qRT-PCR) to quantify the expression of 14-3-3ζ and double immunofluorescence to visualize 14-3-3ζ and apoptosis. We found that, compared with controls, 14-3-3ζ was down-regulated in spina bifida between E12 and E15. Excessive apoptotic cells and low expression of 14-3-3ζ were observed in the dorsal region of spinal cords with spina bifida during the same time period. To initially explore the molecular mechanisms of apoptosis in NTDs, we investigated the expression of microRNA-7 (miR-7), microRNA-375 (miR-375) and microRNA-451 (miR-451), which are known to down-regulate 14-3-3ζ in several different cell types. We also investigated the expression of p53, a molecule that is downstream of 14-3-3ζ and can be down-regulated by it. We discovered that, in contrast to the reduction of 14-3-3ζ expression, the expression of miR-451, miR-375 and p53 increased in spina bifida rat fetuses. CONCLUSIONS/SIGNIFICANCE: These data suggest that the reduced expression of 14-3-3ζ plays a role in the excessive apoptosis that occurs in spina bifida and may be partly regulated by the over-expression of miR-451 and miR-375, and the consequent up-regulation of p53 might further promote apoptosis in spina bifida.